Annexin A6 stimulates the membrane recruitment of p120GAP to modulate Ras and Raf-1 activity

Abstract

Annexin A6 is a calcium-dependent membrane-binding protein that interacts with signalling proteins, including the GTPase-activating protein p120GAP, one of the most important inactivators of Ras. Since we have demonstrated that annexin A6 inhibits EGF-and TPA-induced Ras signalling, we investigated whether modulation of Ras activity by annexin A6 was mediated via altered subcellular localization of p120GAP. First, we exploited our observation that high-density lipoproteins (HDL) can activate the Ras/MAP kinase pathway. Expression of annexin A6 caused a significant reduction in HDLinduced activation of Ras and Raf-1. Annexin A6 promoted membrane binding of p120GAP in vitro, and plasma membrane targeting of p120GAP in living cells, both in a Ca 2 þ -dependent manner, which is consistent with annexin A6 promoting the Ca 2 þ -dependent assembly of p120GAP-Ras at the plasma membrane. We then extended these studies to other cell types and stimuli. Expression of annexin A6 in A431 cells reduced, while RNAi-mediated suppression of annexin A6 in HeLa cells enhanced EGF-induced Ras and Erk activation. Importantly, the enhancement of Ras activation following RNAi-mediated reduction in p120GAP levels was more marked in annexin A6-expressing A431 cells than controls, indicating that the effect of annexin A6 on Ras was mediated via p120GAP. Finally, we demonstrated that annexin A6 promotes plasma membrane targeting of p120GAP in A431 cells in response to a variety of stimuli, resulting in colocalization with H-Ras. These findings demonstrate an important role for annexin A6 in regulating plasma membrane localization of p120GAP and hence Ras activity.

Cholesterol transport from late endosomes to the Golgi regulates t-SNARE trafficking, assembly, and function

“…Importantly, similar results were obtained when anti-SNAP23 or anti–syntaxin-4 staining was compared in A431 wild-type (A431wt) cells, which lack endogenous AnxA6 (Smythe et al , 1994; Grewal et al , 2005), and the stable AnxA6-expressing A431-A6 cell line (Figure S3, A and B). In contrast, stable AnxA6 knockdown HeLa cells (HeLa-A6-KD) showed increased SNAP23 and syntaxin-4 PM localization (Figure S3C).…”

“…Therefore we initially compared the localization of SNAP23 and syntaxin-4 in CHO wild-type (CHOwt) and the well-characterized CHO-A6 cell line (Grewal et al , 2000, 2005; Pons et al , 2001; Rentero et al , 2006; Cubells et al , 2007, 2008). CHOwt cells express low amounts of AnxA6, whereas AnxA6 levels in CHO-A6 cells are similar to AnxA6 expression levels observed in other commonly used cell lines, such as HeLa and NRK (Cubells et al , 2007).…”

“…The generation of the stable AnxA6-overexpressing CHO cell line (CHO-A6) and A431 cell line (A431-A6) has been described in detail (Grewal et al , 2000, 2005; Pons et al , 2001; Cubells et al , 2007; Vila de Muga et al , 2009). To generate AnxA6-overexpressing HuH7, we transfected HuH7 cells with pcDNAanx6 (Grewal et al , 2000) and selected in the presence of 1 mg/ml G418.…”

Differential Regulation of RasGAPs in Cancer

“…37 Moreover, we recently demonstrated that p120GAP-mediated inactivation of epidermal growth factor receptor (EGFR)-induced Ras/MAPK signaling is facilitated by annexin A6 (AnxA6), a member of the annexin protein family. 30,38 Loss of AnxA6 in EGFR-overexpressing and estrogen receptor (ER)-negative breast cancer cells (BCCs) is associated with elevated Ras, Raf, and MAPK activity, and vice versa, upregulation of AnxA6 strongly reduces cell growth of EGFRoverexpressing BCCs. 39,40 Taken together, the potential tumorsuppressive function of each GAP is associated with elevated Ras activity but seems to be restricted to certain cell types, and it is tempting to speculate that specific GAPs control Ras activity in different tissues, but their inhibition is a general mechanism to provoke oncogenic activity and carcinogenesis via elevated wild-type Ras activity.…”

Ras/Mitogen-activated Protein Kinase (MAPK) Signaling Modulates Protein Stability and Cell Surface Expression of Scavenger Receptor SR-BI

“…HDL binding to receptors such as SR-BI 6 activates various cellular processes, including endothelial nitric-oxide synthase activation in endothelial cells (1)(2)(3) and proliferation in smooth muscle cells (4) but also cell surface localization of SR-BI in hepatocytes (5). Downstream targets of HDL include Src family kinases, phospholipase C and D, Ras, phosphatidylinositol 3-kinase (PI3K), Akt, the mitogen-activated protein kinase (MAPK) pathway (Mek1/2 and Erk1/2), and Rac/Rho GTPases (1)(2)(3)(4)(5)(6)(7)(8). Other kinases implicated in HDL-or apoAI-inducible cholesterol transport include protein kinase C (PKC), protein kinase A (PKA), c-Jun N-terminal kinase (JNK), and p38 MAPK (9 -14).…”